The long term objectives of this project are to acquire a molecular-level understanding of the mechanisms that govern cellular control of metabolic pathways. In particular, the aim is to study protein molecules involved in the regulatory process and to examine the molecular basis by which an enzyme can regulate its own activity. The principal investigator has chosen to concentrate on the allosteric enzyme aspartate transcarbamoylase from E. coli, not only because it catalyzes the first reaction in pyrimidine biosynthesis, but also because it exerts control over the entire pathway by a combination of mechanisms that are found in a great many other systems. The diverse physiological functions which are manifest in aspartate transcarbamoylase will help to understand similar functions in many related systems. Of all the metabolic pathways, the biosynthesis of the purines and pyrimidines, which are required in equal amounts for DNA synthesis, are perhaps the most important. Although these pathways have been studied on a macroscopic level, advances in crystallography and biotechnology now make it possible to probe the structure and function of the proteins involved in the control of these pathways at the microscopic level. The understanding of cellular regulation and of the proteins involved in the control process will have a great impact on our grasp of cellular differentiation and, as a consequence of this, cures for diseases such as cancer and birth defects may be found. Previous work on this project has combined the available crystallographic data on the enzyme, with structural and functional studies on mutant enzymes to propose the first molecular model for a concerted allosteric transition in an enzyme. The most recent work suggests direct coupling of catalysis, cooperativity and heterotropic regulation in aspartate transcarbamoylase. It is the mechanism for this direct coupling of homotropic and heterotropic behavior on which the principal investigator intends to concentrate during the next project period. In order to understand how these processes are linked in aspartate transcarbamoylase, he will use a multifaceted approach including functional and structural methods such as: (i) the incorporation of unnatural amino acids to probe function; (ii) analysis of random mutations that are functionally important; (iii) time-resolved low-angle X-ray scattering; (iv) solid-state NMR as a probe of the mechanism of the reaction, and (v) X-ray crystallography of the mutant enzymes.